Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin

摘要

Purpose: Heterozygous mutations in themyocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOCencodes an extracellular protein of unidentified function thatundergoes intracellular endoproteolytic processing in the secretorypathway. It has been described that co-expression of wild-type/mutantmyocilin reduces the secretion of the wild-type protein and that singleexpression of glaucoma myocilin mutants reduces its proteolyticprocessing. However, the effect of wild-type myocilin on mutantmyocilin secretion and how mutant myocilin affects the proteolyticprocessing of wild-type myocilin have not been investigated. We hereinanalyze these two issues. Methods: We modeled the heterozygousstate for 4 missense (E323K, R346T, P370L, D380A) and 1 nonsense(Q368X) myocilin mutants by transiently co-expressing each mutant withthe wild-type protein in HEK-293T cells. Recombinant mutant andwild-type myocilin in both culture media and cellular fractions werequantified by western immunoblot and densitometry. Results: A 24 h transient co-expressionof each myocilin mutant with the wild-type protein elicited anaugmented secretion of the mutant forms from 1.5 fold (D380A) to 5.4fold (E323K). Under such conditions, extracellular mutant myocilinrepresented up to 20% of the total mutant protein. Other than thiseffect, secreted wild-type myocilin significantly decreased from 2.6fold (E323K) to 36 fold (Q368X). When myocilin proteolytic processingwas enhanced (96 hour co-expression) the extracellular amount ofwild-type processed myocilin diminished from approximately 2.1 fold(E323K) to 6.3 fold (P370L). Nonreducing SDS-PAGE indicated thatextracellular myocilin resulting from 24 h co-expression of wild-typemyocilin and each of the 4 missense mutants forms hetero-oligomers andthat glaucoma mutations do not increase the size of myocilinaggregates. Conclusions: Increased extracellularlevels of mutant myocilin expressed in heterozygosis may play arelevant role in glaucoma pathogenesis. This effect is likely theresult of intracellular mutant/wild-type myocilinhetero-oligomerization.